1. The Role of Genes AT5G48150 and AT2G04890 in Arabidopsis thaliana Seed Development Jennifer Huynh June 8, 2006 Honors Collegium 70AL Professor Bob Goldberg

2. Overview

3. What has been already discovered about the genes?

4. The GRAS Family Name derives from the three initially identified members –GAI, RGA, and SCR Functions range from meristem maintenance to regulation of hormone and light signal transduction PAT1 branch plays role in far red light signaling Homologs found in higher plants –Tomato, petunia, lily, rice, barley

5. The GRAS Family Tree

6. The GRAS Family Name derives from the three initially identified members –GAI, RGA, and SCR Functions range from meristem maintenance to regulation of hormone and light signal transduction PAT1 branch plays role in far red light signaling Homologs found in higher plants –Tomato, petunia, lily, rice, barley

7. Gene One: AT5G48150 Scarecrow-like transcription factor 21 (SCL21) Gene Size: 2355 bpProtein Size: 491 aa

8. Gene Two: AT2G04890 Phytochrome A signal transduction 1 (PAT1) Gene Size: 2601 bpProtein Size: 414 aa

9. Where is the gene transcribed into mRNA?

10. mRNA Accumulation: AT5G48150 RT-PCR Results GeneChip Data Control cDNA: 603 bp Control Genomic: 922 bp AT5G48150 Gene: 323 bp

11. mRNA Accumulation: AT2G04890 RT-PCR Results GeneChip Data Control cDNA: 603 bp Control Genomic: 922 bp AT2G04890 Gene: 499 bp

12. Is the upstream region of the gene responsible for the transcription of the gene?

13. Restriction-Digested Plasmid DNA AT5G48150 AT2G04890 Plasmid: 3500 bp Promoter: 3022 bp Gel Bands: 1800 bp, 1200bp Plasmid: ~3500 bp Promoter: 2923 bp Gel Bands: 1600 bp, 1300 bp

14. What happens if the genes are knocked out?

15. Genotyping Insertion Lines AT5G48150 AT2G04890 Wildtype: 1796 bp Salk band: 1740 bp Found band: 1739 bp Wildtype: 1508 bp Salk band: 756 bp Found band: 880 bp

16. Genotyping Insertion Lines AT5G48150 AT2G04890 Wildtype: 1796 bp Salk band: 1740 bp Found band: 1739 bp Wildtype: 1508 bp Salk band: 756 bp Found band: 880 bp

17. AT5G48150: SALK_064220 Wildtype: 1796 base pairs Salk band: 1740 base pairs Found band: 1739 base pairs T-DNA LB RV FW

18. AT2G04890: SALK_146085 Wildtype: 1508 base pairs Salk band: 756 base pairs Found band: 880 base pairs

19. Nomarski Photographs Wildtype Mutant Globular Heart Torpedo Late Torpedo

20. Conclusions No mutant phenotype on flowers, leaves, or siliques for either AT5G48150 or AT2G04890 Possibilities –Genes not vital for seed development –Similar genes exist that compensate –Mutant phenotype not visible in experiments More research is necessary

21. Future Research Perform RT-PCR on homozygous mutants seeds to observe transcription Fuse GFP marker with promoter to observe expression Cross homozygous mutant plants of different knockouts

22. Acknowledgements Bob Goldberg Anhthu Bui Mike Gavino Jonathan Russell Ria Yagnik Brandon Le Xingjun Wang Tomo Kawashima Jessica Luke HC 70AL Classmates